Single domain binding molecule

ABSTRACT

The present invention provides a single domain specific binding molecule having the structure
 
FW1-CDR1-FW2-HV2-FW3a-HV4-FW3b-CDR3-FW4
 
in which the Framework Regions FW1, FW2, FW3a, FW3b, and FW4, the Complementarity Determining Regions CDR1 and CDR3, and the Hypervariable Regions HV2, and HV4 have amino acid sequences as defined which provide a high affinity anti-human serum albumin (HSA) binding domain.

The present invention relates to a single domain specific bindingmolecule derived from an antigen binding protein from cartilaginousfish.

Novel antigen receptor (IgNAR) is a single heavy chain binding domain,devoid of light chain, that exists in the sera of cartilaginous fish(Greenberg et al, Nature, 374 168-173 (1995)). The IgNARs are thereforea class of immunoglobulin-like molecules of the shark immune system thatexist as heavy-chain-only homodimers and bind antigens by their singlevariable domains (VNARs). The distinct structural features of VNARs arethe lack of hydrophobic VH/VL interface residues and the truncation ofCDR2 loop present in conventional immunoglobulin variable domains.

Following shark immunization and/or in vitro selection, VNARs can begenerated as soluble, stable and specific high-affinity monomericbinding proteins of approximately 12 kDa that are amenable to classicphage display selection and screening making them attractive candidatesfor biotherapeutic development (WO 03/014161).

Recent developments in the field of medicine have identified manyantibodies with potentially useful therapeutic applications. However,there are limitations in the current format of these proteins;antibodies being structurally complex multi-domain molecules arerelatively large globular proteins that restrict accessibility toextra-cellular and recessed more cryptic targets. Accordingly, it hasbeen a goal to develop smaller, more stable, specific binding domainswhich can be achieved through reducing the size of the antibody to thebinding domain itself, variations thereof (e.g. scFv, Fab′, Fab, sdAb)or seeking novel scaffolds upon which to engineer target selectivity andaffinity. The challenge in such approaches being that the smaller thedomain, the more rapid its clearance which may be advantageous fordiagnostic imaging, but is far from optimal for the treatment ormanagement of chronic disease. Tailoring the half-life of therapeuticdrugs would negate the requirement for multiple administrations henceminimizing accumulative damage to the patient, increasing patientcompliance and reducing the overall dosing regime which, from acommercial perspective, greatly reduces costs.

Naturally occurring single domain antibodies offer the opportunity toreduce the minimal binding domain further through their inherent lack oflight chain partner. Convergent evolution has resulted in two verydiverse classes of animal developing such domains as an integral part oftheir immune repertoire; the IgNARs from cartilaginous fish and the VHHsor nanobodies from the camelidae (camels, dromedaries and llamas) thatbring great pharmaceutical promise through their stability, solubilityand unique binding loop topography. However with an average molecularmass of 12 to 13 kDa, they are subsequently rapidly cleared in vivo byglomerular filtration.

Significant efforts to address the question of systemic half-lifeextension have included different strategies to counter unfavourablepharmacokinetic properties. Increasing the size of the antibody domainto prevent glomerular clearance has been achieved by increasing thehydrodynamic size via chemical modification of random or directedconjugation to polyethylene glycol (PEG). Other re-formatting strategiessuch as alterations to site-specific glycosylation have shown moderatelyincreased plasma half-life whilst exploitation of the FcRn re-cyclingsystem by molecular Fc fusions have significantly extended circulatingantibody fragment concentration (Pedley et al, British Journal of Cancer70(6), 1126-1130 (1994), Stork et al, The Journal of BiologicalChemistry 283(12), 7804-7812 (2008), Alt et al, FEBS Letters 454(1-2),90-94 (1999)).

Another strategy that hijacks this natural recycling system is the useof serum albumin binding to extend the circulating half-life of smallerproteins or peptides. Albumin is a large (˜67 kDa), abundant serumprotein that plays multiple biological roles in the body such as osmotichaemostasis, fatty acid, lipid and metabolite transfer, metal ionbinding and drug elimination. It has been shown to distribute to regionsof inflammation as illustrated in animal models of arthritis (Fiehn etal, Rheumatology, 43(9), 1097-1105 (2004); Wunder et al, J. Immunol170(9), 4793-4801 (2003)) and to accumulate in proliferatingenvironments such as tumour stroma (Wunder et al, International Journalof Cancer 76(6), 884-890 (1998)).

With a half-life of approximately 19 days in humans, its relativeabundance and unique disease-related distribution profile, serum albuminhas been a target and tool for half-life extension of short-livedsmaller proteins and peptide. This has been achieved through multiplemethods such as chemical linking, association through acylation,molecular fusions and fusion to bacterial albumin binding domains (Smithet al, Bioconjugate Chemistry 12(5), 750-756 (2001), Muller et al, TheJournal of Biological Chemistry, 282(17), 12650-12660 (2007), Stork etal, Protein Eng Des Sel, 20(11), 546-576 (2007)). Peptide display hasyielded multiple different short amino acid sequences that bind withvarying affinities to different albumin species and when fused toantibody fragments, can both increase their half-life and improvebiodistribution (Dennis et al, JBC 277(38), 35035-35043 (2002); Nguyenet al, Protein Eng Des Sel 19(7), 291-297 (2006).). Another proteinbased strategy has been to raise or isolate domain antibodies (dAbs) andcamelid VHH binding domains (nanobodies) against albumin and fuse theseto create albumin binding constructs (Holt et al, Protein Eng Des Sel21(5), 283-288 (2008); Coppieters et al, Arthritis and Rheumatism 54(6),1856-1866 (2006)).

However, there is a continuing need to provide therapeutics with animproved half-life that are active at lower plasma concentrations toavoid the potential for unwanted side-effects at higher dosages. Forsuch therapeutic agents based on immunoglobulin proteins, there is afurther need to provide humanised forms that retain activity.

It has now been found that a particular Variable domain (VNAR) of sharknovel antigen receptor (IgNAR) can provide a high affinity anti-humanserum albumin (HSA) VNAR binding domain. Unusually for this type ofdomain, the interacting residues are not solely within the CDR or HVregions but also include framework residues. The isolation of thisanti-HSA VNAR has significant utility as demonstrated by in vivoefficacy in increasing the sera half-life of a fused unrelated VNARbinding domain. The present invention therefore provides the means fordeveloping bi- or multi-valent therapeutically relevant constructs withextended sera half-life through fusion with the anti-HSA VNAR domain.

According to a first aspect of the invention there is provided a singledomain specific binding molecule having the structureFW1-CDR1-FW2-HV2-FW3a-HV4-FW3b-CDR3-FW4in which the Framework Regions FW1, FW2, FW3a, FW3b, and FW4, theComplementarity Determining Regions CDR1 and CDR3, and the HypervariableRegions HV2, and HV4 have amino acid sequences in which

FW1 comprises TRVDQTPRTATRETGESLTINCVLT (SEQ ID NO: 1),

FW2 comprises TYWYRKNPGS (SEQ ID NO: 2),

FW3a comprises GRYVESVN (SEQ ID NO: 3),

FW3b comprises FSLRIKDLTVADSATYICRA (SEQ ID NO: 4),

FW4 comprises GAGTVLTVN (SEQ ID NO: 5),

CDR1 comprises DTSYPLYS (SEQ ID NO: 6),

CDR3 comprises

(i) MGTNIWTGD (SEQ ID NO: 7),

(ii) MATNIWTGD (SEQ ID NO: 8), MGTDSWTGD (SEQ ID NO:9), MGTNSWTGD (SEQID NO: 10), MSTNIWTGD (SEQ ID NO: 11), ITTDSWTSD (SEQ ID NO: 12),MGANSWTGD (SEQ ID NO: 13), MGTNGWTGD (SEQ ID NO: 14), SDIAMGTYD (SEQ IDNO: 15), ITTHSWSGD (SEQ ID NO: 16), LSTYMEAGD (SEQ ID NO: 17), MDTSAGVVD(SEQ ID NO: 18),

(iii) ESPPICTSQGIAAVTKYYD (SEQ ID NO: 19), YTIHIKLEXH (SEQ ID NO: 20),HAGYGVWNRGLQWRGYDXYD (SEQ ID NO: 21), YTPGREDY (SEQ ID NO: 22),EKGRKGSAITSCRRSSYYD (SEQ ID NO: 23), QSLAISTRSYWYD (SEQ ID NO: 24), or

(iv) GVAGGYCDYALCSSRYAE (SEQ ID NO: 25),

HV2 comprises SNKEQISIS (SEQ ID NO: 26),

HV4 comprises KGTKS (SEQ ID NO: 27),

or a sequence having at least 50% identity thereto, where the amino acidresidue “X” represents glutamine (Q).

Preferred sequences of the invention are shown in FIG. 1(b) as E06, BB11and E06 with a poly-histidine C-terminal sequence (E06-AAA-6×His), or asequence having at least 50% identity thereto. In a comparison of E06(SEQ ID NO: 46) and BB11 (SEQ ID NO: 85), there are 28 out of 103 aminoacid residues that are different, so the sequences are 27% different andhave a degree of homology of 73%.

Where “X” is Q, then sequences YTIHIKLEXH (SEQ ID NO: 20) andHAGYGVWNRGLQWRGYDXYD (SEQ ID NO: 21) are YTIHIKLEQH (SEQ ID NO: 20) andHAGYGVWNRGLQWRGYDQYD (SEQ ID NO: 21) respectively.

The single domain specific binding molecule of the present invention isbased on a single variable domain (VNAR) of an IgNAR immunoglobulin-likemolecule. The single domain specific binding molecule suitably comprisesthe following domains:FW1-CDR1-FW2-HV2-FW3a-HV4-FW3b-CDR3-FW4

in which FW is a Framework Region, CDR is a Complementarity DeterminingRegion, and HV is a Hypervariable Region. In common with other VNARmolecules, there is no CDR2 region as in a mammalian antibody variable(V) domain.

In one embodiment of the invention, there is provided a single domainspecific binding molecule having the structureFW1-CDR1-FW2-HV2-FW3a-HV4-FW3b-CDR3-FW4

in which

FW1, FW2, FW3a, FW3b and FW4 are Framework Regions having the amino acidsequences TRVDQTPRTATRETGESLTINCVLT (SEQ ID NO: 1), TYWYRKNPGS (SEQ IDNO: 2), GRYVESVN (SEQ ID NO: 3),

FSLRIKDLTVADSATYICRA (SEQ ID NO: 4), and GAGTVLTVN (SEQ ID NO: 5)respectively;

CDR1 and CDR3 are Complementarity Determining Regions having the aminoacid sequences DTSYPLYS (SEQ ID NO: 6), and MGTNIWTGD (SEQ ID NO: 7) orGVAGGYCDYALCSSRYAE (SEQ ID NO: 25) respectively;

HV2 and HV4 are Hypervariable Regions having the amino acid sequencesSNKEQISIS (SEQ ID NO: 26) and KGTKS (SEQ ID NO: 27) respectively;

or a sequence having at least 50% identity thereto. An example of asingle domain specific binding molecule having the specific sequencedefined where CDR1 and CDR3 are DTSYPLYS (SEQ ID NO: 6), and MGTNIWTGD(SEQ ID NO: 7) is sequence E06.

In one embodiment of the invention, there is provided a single domainspecific binding molecule having the structureFW1-CDR1-FW2-HV2-FW3a-HV4-FW3b-CDR3-FW4

in which

FW1, FW2, FW3a, FW3b and FW4 are Framework Regions having the amino acidsequences

TRVDQSPSSLSASVGDRVTITCVLT (SEQ ID NO: 28), TYWYRKNPGS (SEQ ID NO: 2),GRYSESVN (SEQ ID NO: 30),

FTLTISSLQPEDFATYYCRA (SEQ ID NO: 31) and GAGTKVEIK (SEQ ID NO: 32)respectively

CDR1 and CDR3 are Complementarity Determining Regions having the aminoacid sequences DTSYPLYS (SEQ ID NO: 6) and MATNIWTGD (SEQ ID NO: 8)respectively;

HV2 and HV4 are Hypervariable Regions having the amino acid sequencesSNKEQISIS (SEQ ID NO: 26) and KGTKS (SEQ ID NO: 27) respectively;

or a sequence having at least 50% identity thereto. The single domainspecific binding molecule having the above specific sequence is BB11.

In another embodiment of the invention, the Framework Regions FW1, FW2,FW3a, FW3b and FW4 of the single domain specific binding molecule havethe amino acid sequences TRVDQSPSSLSASVGDRVTITCVLT (SEQ ID NO: 28),TYWYQQKPGS (SEQ ID NO: 29), GRYSESVN (SEQ ID NO: 30),FTLTISSLQPEDFATYYCRA (SEQ ID NO: 31) and GAGTKVEIK (SEQ ID NO: 32)respectively.

Specific binding molecules of this aspect of the invention may havesequences as set out in FIG. 1(a): P2_A03, P2_C06, P2_E06, H08, P3_A03,P3_A08, P3_A12, P3_B08, P3_B09, P3_D03, P3_D05, P3_D10, P3_D11, E06,P3_E07, P3_F03, P3_F11, and P3_G10, or a sequence having at least 50%identity thereto.

In another aspect of the invention, the specific binding molecule mayhave a sequence as shown in FIG. 1(c): P0_02_B12, P0_05_E09, P1_09_C07,P1_08_E06, P1_07_B06, P1_10_A01, P1_07_G11, P1_10_D05, P1_09_C11,P1_10_A02, P1_10_C03, and P1_10_C11, or a sequence having at least 50%identity thereto. The sequences shown in FIG. 1(c) also relate toanti-HSA binding domains isolated from a pre-selected phage displaylibrary.

In another aspect of the invention, the specific binding molecule mayhave a sequence as shown in FIG. 2(a), 2(b) or 2(c), in particularsequences E06, BB11, 5A7, 5A7-IVabc, huE06 v1.1, huH08 v1.1, huE06 v1.2,huE06 v1.3, huE06 v1.4, huE06 v1.5, huE06 v1.6, huE06 v1.7, huE06 v1.8,huE06 v1.9, huE06 v1.10, AC9, AD4, AG11, AH7, BA11, BB10, BC3, BD12,BE4, or BH4, or a sequence having at least 50% identity thereto. Thesequences huE06 v1.10 and BB11 therefore share the same framework anddiffer by one amino acid residue in CDR3.

In one embodiment of this aspect of the invention, the CDR3 regioncomprises

(i) MGTNIWTGD (SEQ ID NO: 7),

(ii) GVAGGYCDYALCSSRYAE (SEQ ID NO: 25),

(iii) ESPPICTSQGIAAVTKYYD (SEQ ID NO: 19), YTIHIKLEXH (SEQ ID NO: 20),HAGYGVWNRGLQWRGYDXYD (SEQ ID NO: 21), YTPGREDY (SEQ ID NO: 22),EKGRKGSAITSCRRSSYYD (SEQ ID NO: 23), QSLAISTRSYWYD (SEQ ID NO: 24), or

(iv) ITTDSWTSD (SEQ ID NO: 12), MGANSWTGD (SEQ ID NO: 13), MGTNGWTGD(SEQ ID NO: 14), SDIAMGTYD (SEQ ID NO: 15), ITTHSWSGD (SEQ ID NO: 16),LSTYMEAGD (SEQ ID NO: 17), MDTSAGVVD (SEQ ID NO: 18),

or a sequence having at least 50% identity thereto. The amino acidresidue “X” represents glutamine (Q).

The term “protein” in this text means, in general terms, a plurality ofamino acid residues joined together by peptide bonds. It is usedinterchangeably and means the same as peptide, oligopeptide, oligomer orpolypeptide, and includes glycoproteins and derivatives thereof. Theterm “protein” is also intended to include fragments, analogues,variants and derivatives of a protein wherein the fragment, analogue,variant or derivative retains essentially the same biological activityor function as a reference protein. Examples of protein analogues andderivatives include peptide nucleic acids, and DARPins (Designed AnkyrinRepeat Proteins).

The fragment, analogue, variant or derivative of the protein as definedin this text, may be at least 25 preferably 30 or 40, or up to 50 or100, or 60 to 120 amino acids long, depending on the length of theoriginal protein sequence from which it is derived. A length of 90 to120, 100 to 110 amino acids may be convenient in some instances.

The fragment, derivative, variant or analogue of the protein may be (i)one in which one or more of the amino acid residues are substituted witha conserved or non-conserved amino acid residue (preferably, a conservedamino acid residue) and such substituted amino acid residue may or maynot be one encoded by the genetic code, or (ii) one in which one or moreof the amino acid residues includes a substituent group, or (iii) one inwhich the additional amino acids are fused to the mature polypeptide,such as a leader or auxiliary sequence which is employed forpurification of the polypeptide. Such fragments, derivatives, variantsand analogues are deemed to be within the scope of those skilled in theart from the teachings herein.

In certain preferred embodiments of the invention, the single domainspecific binding molecule has an amino acid sequence selected from thegroup consisting of VNAR domains shown in FIG. 1(a), in which FW1comprises residues 1 to 25 CDR1 comprises residues 26 to 33, FW2comprises residues 34 to 43, HV2 comprises residues 44 to 52, FW3acomprises residues 53 to 60, HV4 comprises residues 61 to 65, FW3bcomprises residues 66 to 85, CDR3 comprises residues 86 to 94 and FW4comprises residues 95 to 103, or any combination thereof.

In certain preferred embodiments of the invention, the single domainspecific binding molecule has an amino acid sequence selected from thegroup consisting of VNAR domains shown in FIG. 1(c), in which FW1comprises residues 1 to 27, CDR1 comprises residues 28 to 33, FW2comprises residues 34 to 43, HV2 comprises residues 44 to 52, FW3acomprises residues 53 to 60, HV4 comprises residues 61 to 65, FW3bcomprises residues 66 to 85, CDR3 comprises residues 86 to 105 and FW4comprises residues 106 to 113, or any combination thereof, in which oneor more residues may be absent from CDR3, suitably 1 to 12 residues,with respect to the consensus sequence.

In certain preferred embodiments of the invention, the single domainspecific binding molecule has an amino acid sequence selected from thegroup consisting of VNAR domains shown in FIG. 2(a) or FIG. 2(b), or anycombination thereof.

In one embodiment of the invention, the single domain specific bindingmolecule is an amino acid sequence as shown in FIG. 2(a) or 2(b) or anyvariant, analogue, derivative or fragment thereof, including a sequencehaving 50% identity thereto. Such sequences may be selected from thegroup consisting of: huE06 v1.1, huH08 v1.1, huE06 v1.2, huE06 v1.3,huE06 v1.4, huE06 v1.5, huE06 v1.6, huE06 v1.7, huE06 v1.8, huE06 v1.9,or huE06 v1.10, as shown in FIG. 2 or any combination thereof, or asequence having 50% identity thereto. The single domain specific bindingmolecule known as E06 is suitably an isolated protein or peptidesequence comprising the nucleotide and amino acid sequences shown inFIG. 1(b).

In other embodiments of the invention, the specific binding molecule maybe huE06 v1.10 as shown in FIG. 2(c) or any variant, analogue,derivative or fragment thereof, including a sequence having 50% identitythereto.

In one embodiment of the invention, the single domain specific bindingmolecule is humanized. The degree of humanization may be designedaccording to the antibody properties required, including antibodyaffinity. A suitable degree of humanisation may be 50%, or 55%, 60%,65%, 70%, 75%, 80%, 85%, 90% or 95% homology to the corresponding humangermline sequence for a Framework Region or a CDR Region specific to theantigen epitope of interest. For example, sequence huE06 v1.10 isapproximately 60% humanized with respect to the native E06 sequence.

The single domain specific binding molecule may comprise additionalN-terminal or C-terminal sequences which are cleaved off prior to usewhich may assist in purification and/or isolation during processes forthe production of the molecule as described herein. For example,(Ala)₃(His)₆ (SEQ ID NO: 90) at the C-terminal end of the molecule.

In one embodiment, the framework FW1 region may comprise an amino acidsequence of TRVDQSPSSLSASVGDRVTITCVLT (SEQ ID NO: 28), the framework FW2region may comprise an amino acid sequence of TYWYQQKP (SEQ ID NO: 91),the hypervariable HV2 region may comprise an amino acid sequence ofSNKEQISIS (SEQ ID NO: 26), the framework FW3a region may comprise anamino acid sequence of RYSESVN (SEQ ID NO: 92), the hypervariable HV4region may comprise an amino acid sequence of KGTKS (SEQ ID NO: 27), theframework FW3b region may comprise an amino acid sequence ofFTLTISSLQPEDFATYYC (SEQ ID NO: 93), and the framework FW4 region maycomprise an amino acid sequence of GAGTKVEIK (SEQ ID NO: 32), orsequences having 50% identity thereto.

Single domain specific binding molecules of the invention may thereforebe constructed of any of the amino acid sequences for the variousregions disclosed herein according to the basic structure:FW1-CDR1-FW2-HV2-FW3a-HV4-FW3b-CDR3-FW4

The domain described herein as E06 was isolated from an immunized sharklibrary, has picomolar high affinity, and high selectivity for humanalbumin and improves the pharmacokinetics (PK) of a companion “dummy”protein (fused to both N- and C-termini) across three species (mouse,rat and monkey) giving a predicted half-life of 19 days in man.

Also included within the invention are variants, analogues, derivativesand fragments having the amino acid sequence of the protein in whichseveral e.g. 5 to 10, or 1 to 5, or 1 to 3, 2, 1 or no amino acidresidues are substituted, deleted or added in any combination.Especially preferred among these are silent substitutions, additions anddeletions, which do not alter the properties and activities of theprotein of the present invention. Also especially preferred in thisregard are conservative substitutions where the properties of a fusionprotein of the present invention are preserved in the variant formcompared to the original form.

Variants therefore include fusion proteins comprising a single domainspecific binding molecule according to the first aspect of theinvention.

The fusion protein may comprise a single domain specific bindingmolecule of the present invention fused to a heterologous peptide orprotein sequence providing a structural element to the fusion protein.In other embodiments, the fusion protein may comprise a single domainspecific binding molecule of the present invention fused with a moleculehaving biological activity. The molecule may be a peptide or proteinsequence, or another biologically active molecule. Such a conjugate maytherefore comprise a non-protein biologically active molecule also.

For example, the single domain specific binding molecule may be fused toa heterologous peptide sequence which may be a poly-amino acid sequence,for example a plurality of histidine residues or a plurality of lysineresidues (suitably 2, 3, 4, 5, or 6 residues), or an immunoglobulindomain (for example an Fc domain).

References to heterologous peptides sequences include sequences fromother mammalian species, such as murine and human and any heterologouspeptides sequences originated from other VNAR domains.

Where the fusion protein comprises a single domain specific bindingmolecule of the present invention fused with a molecule havingbiological activity, biologically active moiety may be a peptide orprotein having biological activity such as an enzyme, immunoglobulin,cytokine or a fragment thereof. Alternatively, the biologically activemolecule may be an antibiotic, an anti-cancer drug, an NSAID, a steroid,an analgesic, a toxin or other pharmaceutically active agent.Anti-cancer drugs may include cytotoxic or cytostatic drugs. Specificexamples are described in greater detail below in relation topharmaceutical compositions comprising such fusion proteins.

In some embodiments, the fusion protein may comprise a single domainspecific molecule of the invention fused to another immunoglobulinvariable or constant region, or another single domain specific moleculeof the invention. In other words, fusions of the single domain specificbinding molecules of the invention of variable length, e.g. dimers,trimers, tetramers, or higher multimer (i.e. pentamers, hexamers,heptamers octamers, nonamers, or decamers, or greater). In specificembodiments this can be represented as a multimer of monomer VNARsubunits.

In fusion proteins of the present invention, the single domain specificbinding molecule may be directly fused or linked via a linker moiety tothe other elements of the fusion protein. The linker may be a peptide,peptide nucleic acid, or polyamide linkage. Suitable peptide linkers mayinclude a plurality of amino acid residues, for example, 4, 5, 6, 7, 8,9, 10, 15, 20 or 25 amino acids, such as (Gly)₄ (SEQ ID NO: 94), (Gly)₅(SEQ ID NO: 95), (Gly)₄Ser (SEQ ID NO: 96), (Gly)₄(Ser)(Gly)₄ (SEQ IDNO: 97), or combinations thereof or a multimer thereof (for example adimer, a trimer, or a tetramer, or greater). For example, a suitablelinker may be (GGGGS)₃ (SEQ ID NO: 98). Alternative linkers include(Ala)₃(His)₆ (SEQ ID NO: 90) or multimers thereof. Also included is asequence which has at least 50%, 60%, 70%, 80%, 90%, 95% or 99%identity, using the default parameters of the BLAST computer programprovided by HGMP, thereto.

An example of a variant of the present invention is a fusion protein asdefined above, apart from the substitution of one or more amino acidswith one or more other amino acids. The skilled person is aware thatvarious amino acids have similar properties. One or more such aminoacids of a substance can often be substituted by one or more other suchamino acids without interfering with or eliminating a desired activityof that substance. Such substitutions may be referred to as“non-conservative” amino acid substitutions.

Thus the amino acids glycine, alanine, valine, leucine and isoleucinecan often be substituted for one another (amino acids having aliphaticside chains). Of these possible substitutions it is preferred thatglycine and alanine are used to substitute for one another (since theyhave relatively short side chains) and that valine, leucine andisoleucine are used to substitute for one another (since they havelarger aliphatic side chains which are hydrophobic). Other amino acidswhich can often be substituted for one another include: phenylalanine,tyrosine and tryptophan (amino acids having aromatic side chains);lysine, arginine and histidine (amino acids having basic side chains);aspartate and glutamate (amino acids having acidic side chains);asparagine and glutamine (amino acids having amide side chains); andcysteine and methionine (amino acids having sulphur containing sidechains). Substitutions of this nature are often referred to as“conservative” or “semi-conservative” amino acid substitutions.

Amino acid deletions or insertions may also be made relative to theamino acid sequence for the fusion protein referred to above. Thus, forexample, amino acids which do not have a substantial effect on theactivity of the polypeptide, or at least which do not eliminate suchactivity, may be deleted. Such deletions can be advantageous since theoverall length and the molecular weight of a polypeptide can be reducedwhilst still retaining activity. This can enable the amount ofpolypeptide required for a particular purpose to be reduced—for example,dosage levels can be reduced. Amino acid insertions relative to thesequence of the fusion protein above can also be made. This may be doneto alter the properties of a substance of the present invention (e.g. toassist in identification, purification or expression, as explained abovein relation to fusion proteins). Amino acid changes relative to thesequence for the fusion protein of the invention can be made using anysuitable technique e.g. by using site-directed mutagenesis.

It should be appreciated that amino acid substitutions or insertionswithin the scope of the present invention can be made using naturallyoccurring or non-naturally occurring amino acids. Whether or not naturalor synthetic amino acids are used, it is preferred that only L-aminoacids are present.

A protein according to the invention may have additional N-terminaland/or C-terminal amino acid sequences. Such sequences can be providedfor various reasons, for example, glycosylation.

The term “fusion protein” in this text means, in general terms, one ormore proteins joined together by chemical means, including hydrogenbonds or salt bridges, or by peptide bonds through protein synthesis orboth.

“Identity” as known in the art is the relationship between two or morepolypeptide sequences or two or more polynucleotide sequences, asdetermined by comparing the sequences. In the art, identity also meansthe degree of sequence relatedness (homology) between polypeptide orpolynucleotide sequences, as the case may be, as determined by the matchbetween strings of such sequences. While there exist a number of methodsto measure identity between two polypeptide or two polynucleotidesequences, methods commonly employed to determine identity are codifiedin computer programs. Preferred computer programs to determine identitybetween two sequences include, but are not limited to, GCG programpackage (Devereux, et al., Nucleic acids Research, 12, 387 (1984),BLASTP, BLASTN, and FASTA (Atschul et al., J. Molec. Biol. 215, 403(1990).

Preferably, the amino acid sequence of the protein has at least 50%identity, using the default parameters of the BLAST computer program(Atschul et al., J. Mol. Biol. 215, 403-410 (1990) provided by HGMP(Human Genome Mapping Project), at the amino acid level, to the aminoacid sequences disclosed herein.

More preferably, the protein sequence may have at least 55%, 60%, 65%,66%, 67%, 68%, 69%, 70%, 75%, 80%, 85%, 90% and still more preferably95% (still more preferably at least 96%, 97%, 98% or 99%) identity, atthe nucleic acid or amino acid level, to the amino acid sequences asshown herein.

The protein may also comprise a sequence which has at least 50%, 55%,60%, 65%, 66%, 67%, 68%, 69%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%,98%, or 99% identity with a sequence disclosed herein, using the defaultparameters of the BLAST computer program provided by HGMP, thereto.

According to a second aspect of the invention, there is provided anucleic acid encoding a single domain specific binding molecule of thesecond aspect of the invention.

According to a third aspect of the invention, there is provided anucleic acid construct comprising a nucleic acid of the second aspect ofthe invention.

The term “nucleic acid construct” generally refers to any length ofnucleic acid which may be DNA, cDNA or RNA such as mRNA obtained bycloning or produced by chemical synthesis. The DNA may be single ordouble stranded. Single stranded DNA may be the coding sense strand, orit may be the non-coding or anti-sense strand. For therapeutic use, thenucleic acid construct is preferably in a form capable of beingexpressed in the subject to be treated.

The nucleic acid construct of the third aspect of the invention may bein the form of a vector, for example, an expression vector, and mayinclude, among others, chromosomal, episomal and virus-derived vectors,for example, vectors derived from bacterial plasmids, frombacteriophage, from transposons, from yeast episomes, from insertionelements, from yeast chromosomal elements, from viruses such asbaculo-viruses, papova-viruses, such as SV40, vaccinia viruses,adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses,and vectors derived from combinations thereof, such as those derivedfrom plasmid and bacteriophage genetic elements, such as cosmids andphagemids. Generally, any vector suitable to maintain, propagate orexpress nucleic acid to express a polypeptide in a host, may be used forexpression in this regard.

The nucleic acid construct of the third aspect of the inventionpreferably includes a promoter or other regulatory sequence whichcontrols expression of the nucleic acid. Promoters and other regulatorysequences which control expression of a nucleic acid have beenidentified and are known in the art. The person skilled in the art willnote that it may not be necessary to utilise the whole promoter or otherregulatory sequence. Only the minimum essential regulatory element maybe required and, in fact, such elements can be used to constructchimeric sequences or other promoters. The essential requirement is, ofcourse, to retain the tissue and/or temporal specificity. The promotermay be any suitable known promoter, for example, the humancytomegalovirus (CMV) promoter, the CMV immediate early promoter, theHSV thymidinekinase, the early and late SV40 promoters or the promotersof retroviral LTRs, such as those of the Rous Sarcoma virus (RSV) andmetallothionine promoters such as the mouse metallothionine-I promoter.The promoter may comprise the minimum comprised for promoter activity(such as a TATA elements without enhancer elements) for example, theminimum sequence of the CMV promoter. Preferably, the promoter iscontiguous to the nucleic acid sequence.

As stated herein, the nucleic acid construct of the third aspect of theinvention may be in the form of a vector. Vectors frequently include oneor more expression markers which enable selection of cells transfected(or transformed) with them, and preferably, to enable a selection ofcells containing vectors incorporating heterologous DNA. A suitablestart and stop signal will generally be present.

The vector may be any suitable expression vector, such as pET. Thevector may include such additional control sequences as desired, forexample selectable markers (e.g. antibiotic resistance, fluorescence,etc.), transcriptional control sequences and promoters, includinginitiation and termination sequences. The promoter may be any suitablepromoter for causing expression of the protein encoded by a nucleic acidsequence of the invention, e.g. a CMV promoter, human phosphoglyceratekinase (hPGK) promoter.

According to a fourth aspect of the invention, there is provided a hostcell comprising a vector according to the third aspect of the invention.Representative examples of appropriate host cells for expression of thenucleic acid construct of the invention include virus packaging cellswhich allow encapsulation of the nucleic acid into a viral vector;bacterial cells, such as Streptococci, Staphylococci, E. coli,Streptomyces and Bacillus subtilis; single cells, such as yeast cells,for example, Saccharomyces cerevisiae, and Aspergillus cells; insectcells such as Drosophila S2 and Spodoptera Sf9 cells, animal cells suchas CHO, COS, C127, 3T3, PHK.293, and Bowes Melanoma cells and othersuitable human cells; and plant cells e.g. Arabidopsis thaliana.Suitably, the host cell is a eukaryotic cell, such as a CHO cell or aHEK293 cell.

Introduction of an expression vector into the host cell can be achievedby calcium phosphate transfection, DEAE-dextran mediated transfection,microinjection, cationic—lipid-mediated transfection, electroporation,transduction, scrape loading, ballistic introduction, infection or othermethods. Such methods are described in many standard laboratory manuals,such as Sambrook et al, Molecular Cloning, a Laboratory Manual, SecondEdition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.(1989).

Mature proteins can be expressed in host cells, including mammaliancells such as CHO cells, yeast, bacteria, or other cells under thecontrol of appropriate promoters. Cell-free translation systems can beemployed to produce such proteins using RNAs derived from the nucleicacid construct of the third aspect of the present invention. Appropriatecloning and expression vectors for use with prokaryotic and eukaryotichosts are described by Sambrook et al, Molecular Cloning, a LaboratoryManual, Second Edition, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. (1989).

According to a fifth aspect of the invention, there is provided aprocess for the production of a single domain specific binding moleculeof the first aspect of the invention, comprising the step of expressinga nucleic acid sequence encoding said molecule in a suitable host cellas defined herein.

Proteins can be recovered and purified from recombinant cell cultures bywell-known methods including ammonium sulphate or ethanol precipitation,acid extraction, anion or cation exchange chromatography,phosphocellulose chromatography, hydrophobic interaction chromatography,affinity chromatography, hydroxyapatite chromatography, lectin and/orheparin chromatography. For therapy, the nucleic acid construct e.g. inthe form of a recombinant vector, may be purified by techniques known inthe art, such as by means of column chromatography as described inSambrook et al, Molecular Cloning, a Laboratory Manual, Second Edition,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989).

This aspect of the invention therefore extends to processes forpreparing a fusion protein of the first aspect of the inventioncomprising production of the fusion protein recombinantly by expressionin a host cell, purification of the expressed fusion protein andassociation of the pharmaceutically active agent to the purified fusionprotein by means of peptide bond linkage, hydrogen or salt bond orchemical cross-linking. In some embodiments of this aspect of theinvention where the pharmaceutically active agent is a peptide, thefusion protein could be prepared using hydrogen or salt bonds where thepeptide is capable or multimerisation, for example dimerisation ortrimerisation.

According to an sixth aspect of the invention, there is provided apharmaceutical composition of a single domain specific binding moleculeof the first aspect of the invention. Such compositions include fusionproteins comprising said single domain specific binding molecules.

The pharmaceutical composition may comprise a single domain specificbinding molecule of the present invention fused to a therapeutic proteinor a fragment thereof, or any other pharmaceutically active agent(non-protein based) or other chemical compound or polymer as describedabove. The conjugate may comprise therefore a biologically active agentfused to a single domain specific binding molecule of the invention.

The therapeutic protein may be a hormone, a growth factor (e.g. TGFβ,epidermal growth factor (EGF), platelet derived growth factor (PDGF),nerve growth factor (NGF), colony stimulating factor (CSF), hepatocytegrowth factor, insulin-like growth factor, placenta growth factor); adifferentiation factor; a blood clotting factor ( ); for example FactorVIla, Factor VIII, Factor IX, VonWillebrand Factor or Protein C) oranother protein from the blood coagulation cascade (for exampleantithrombin); a cytokine e.g. an interleukin, (e.g. IL1, IL-2, IL-3,IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14,IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24,IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32 or IL-33 or aninterferon (e.g. IFN-α, IFN-β and IFN-γ), tumour necrosis factor (TNF),IFN-γ inducing factor (IGIF), a bone morphogenetic protein (BMP, e.g.BMP-1, BMP-2, BMP-3, BMP-4, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8, BMP-9,BMP10, BMP-11, BMP-12, BMP-13); an interleukin receptor antagonist (e.g.IL-1ra, IL-1RII); a chemokine (e.g. MIPs (Macrophage InflammatoryProteins) e.g. MIP1α and MIP1β; MCPs (Monocyte Chemotactic Proteins)e.g. MCP1, 2 or 3; RANTES (regulated upon activation normal T-cellexpressed and secreted)); a trophic factor; a cytokine inhibitor; acytokine receptor; an enzyme, for example a free-radical scavengingenzyme e.g. superoxide dismutase or catalase or a pro-drug convertingenzyme (e.g. angiotensin converting enzyme, deaminases, dehydrogenases,reductases, kinases and phosphatases); a peptide mimetic; a proteaseinhibitor; a tissue inhibitor of metalloproteinases (TIMPs e.g. TIMP1,TIMP2, TIMP3 or TIMP4) or a serpin (inhibitors of serine proteases).

In other embodiments of the invention, the therapeutic protein may be anantibody, or a engineered fragment thereof, including Fab, Fc, F(ab′)₂(including chemically linked F(ab′)₂ chains), Fab′, scFv (includingmultimer forms thereof, i.e. di-scFv, or tri-scFv), sdAb, or BiTE(bi-specific T-cell engager). Antibody fragments also include variabledomains and fragments thereof, as well as other VNAR type fragments(IgNAR molecules).

The pharmaceutically active agent in such fusion proteins may be atherapeutic compound, e.g. anti-inflammatory drug (e.g. a non-steroidalanti-inflammatory drug), cytotoxic agent (e.g. a toxin, such as choleratoxin, or a radionuclide comprising a radioactive element fortherapeutic or diagnostic use), cytostatic agent, or antibiotic. Theother chemical compound or polymer may be a substance suitable to extendthe half-life of the fusion protein in vivo. Suitable conjugates for usein such fusion proteins include polyethylene glycol (PEG) and/orcyclodextrin.

The pharmaceutical composition may be composed of a number of singledomain specific binding molecules of the invention, for example dimers,trimers, or higher order multimers, i.e. 2, 3, 4, 5, 6, 7, or 8-mers,fused to the therapeutic protein.

The fusion of the single domain specific binding molecules of theinvention to the therapeutic protein may at any convenient site on theprotein and may be N-, C- and/or N-/C-terminal fusion(s). In oneembodiment of the invention, the fusion of the single domain specificbinding molecules of the invention is to both the N- and C-terminals ofa therapeutic protein. Such fusion proteins may be prepared by anysuitable route, including by recombinant techniques by expression inhost cell or cell-free systems, as well as by chemical synthetic routes.Conjugates of non-protein biologically active agents or other chemicalcompounds or polymers may be achieved by any suitable chemical syntheticor biosynthetic (e.g. enzymatic) route.

Such compositions may comprise any suitable and pharmaceuticallyacceptable carrier, diluent, adjuvant or buffer solution. Thecomposition may comprise a further pharmaceutically active agent. Suchcarriers may include, but are not limited to, saline, buffered saline,dextrose, liposomes, water, glycerol, ethanol and combinations thereof.

Such compositions may comprise a further pharmaceutically active agentas indicated. The additional agents may be therapeutic compounds, e.g.anti-inflammatory drugs, cytotoxic agents, cytostatic agents orantibiotics. Such additional agents may be present in a form suitablefor administration to patient in need thereof and such administrationmay be simultaneous, separate or sequential. The components may beprepared in the form of a kit which may comprise instructions asappropriate.

The pharmaceutical compositions may be administered in any effective,convenient manner effective for treating a patient's disease including,for instance, administration by oral, topical, intravenous,intramuscular, intranasal, or intradermal routes among others. Intherapy or as a prophylactic, the active agent may be administered to anindividual as an injectable composition, for example as a sterileaqueous dispersion, preferably isotonic.

For administration to mammals, and particularly humans, it is expectedthat the daily dosage of the active agent will be from 0.01 mg/kg bodyweight, typically around 1 mg/kg, 2 mg/kg or up to 4 mg/kg. Thephysician in any event will determine the actual dosage which will bemost suitable for an individual which will be dependent on factorsincluding the age, weight, sex and response of the individual. The abovedosages are exemplary of the average case. There can, of course, beinstances where higher or lower dosages are merited, and such are withinthe scope of this invention.

According to a seventh aspect of the invention, there is provided asingle domain specific binding molecule of the first aspect of theinvention for use in medicine. This aspect of the invention thereforeextends to the use of such of a single domain specific binding moleculeof the first aspect in the manufacture of a medicament for the treatmentof a disease in a patient in need thereof. A single domain specificbinding protein of the invention can therefore be used to prepare afusion protein comprising such a specific binding molecule as definedabove in relation to pharmaceutical compositions of the invention.

Such uses also embrace methods of treatment of diseases in patients inneed of treatment comprising administration to the patient of atherapeutically effective dosage of a pharmaceutical composition asdefined herein comprising a single domain specific binding molecule ofthe first aspect of the invention.

As used herein, the term “treatment” includes any regime that canbenefit a human or a non-human animal. The treatment of “non-humananimals” in veterinary medicine extends to the treatment of domesticanimals, including horses and companion animals (e.g. cats and dogs) andfarm/agricultural animals including members of the ovine, caprine,porcine, bovine and equine families. The treatment may be a therapeutictreatment in respect of any existing condition or disorder, or may beprophylactic (preventive treatment). The treatment may be of aninherited or an acquired disease. The treatment may be of an acute orchronic condition. The treatment may be of a condition/disorderassociated with inflammation and/or cancer. The single domain specificbinding molecules of the invention may be used in the treatment of adisorder, including, but not limited to osteoarthritis, scleroderma,renal disease, rheumatoid arthritis, inflammatory bowel disease,multiple sclerosis, atherosclerosis, or any inflammatory disease.

The specific binding molecules of the present invention may also be usedto investigate the nature of a disease condition in a patient. Thesingle domain specific binding molecules may be used to prepare imagesof sites of disease in the body of a subject using imaging techniquessuch as X-ray, gamma-ray, or PET scanning, or similar. The invention maytherefore extend to a method of imaging a site of disease in a subject,comprising administration of a suitably detectably labelled singledomain specific binding molecule to a subject and scanning the subject'sbody subsequently. Alternatively, administration of said molecules to asubject may provide for a test result by analysing a sample from thesubject following administration of the molecule. Such embodiments mayinclude a method of diagnosis of a disease or medical condition in asubject comprising administration of a single domain specific bindingmolecule of the invention. The embodiment includes methods of detectinga site of disease or medical condition and specific binding molecules ofthe present invention for use in such methods.

Such uses of the specific binding molecules of the invention thereforeextends to uses in research in cell culture in vitro where the specificbinding molecule is used as a tool to investigate cellular processesand/or behaviour.

The present invention is based on an unexpected immune response based onIgNAR from a dogfish (Squalus acanthias) challenged with human serumalbumin resulting in the isolation of a specific variable domain from anaturally evolved IgNAR. This invention shows for the first time thatIgNAR forms part of the adaptive immune response of these animals thatare approximately 200M years distinct in evolutionary terms from Nursesharks who have previously been shown to respond to immunization (Dooleyet al, Mol Immunol 40(1) 25-33 (2003). A high affinity, highly selectivedomain against HSA known as E06 was isolated after screening againsttarget. Unusually for VNAR domain, the interface with target was planaras elucidated by the crystal structure. Unexpectedly E06 is tolerant ofboth N-terminal, C-terminal and dual partner protein fusions and has apredicted half-life (as a fusion) of 19 days in man as predicted byserum half-life analyses across three species. The isolation of thisanti-HSA VNAR therefore has significant utility as a means fordeveloping bi- or multi-valent therapeutically relevant constructs withextended sera half-life through fusion with the anti-HSA VNAR domain.

All preferred features of the second and subsequent aspects of theinvention are as for the first aspect mutatis mutandis.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A to 1C show the amino acid sequences of the natural specificbinding domains of the invention: a) an alignment of the anti-HSAbinding domains isolated after round 2 and 3 of selection (SEQ ID NOs:33-50); b) (i) the amino acid of the specific binding domain E06 (SEQ IDNO: 46); (ii) a comparison of sequence E06 (SEQ ID NO: 46) and BB11 (SEQID NO: 85); and (iii) sequence E06 with a 3×Ala-6×HIS tag (AAA-6×His tagis in italic) (SEQ ID NO: 51); c) sequence alignment of anti-HSA bindingdomains isolated from the pre-selected phage display library (SEQ IDNOs: 52-64). (Shading (with solid grey) indicates residues that differfrom the consensus in FIG. 1(a) and 1(c)).

FIGS. 2A to 2C: (a) shows structural sequence alignment of VNAR E06 (SEQID NO: 46) and its humanized variants of the invention (SEQ ID NOs:65-74) including human germline V-kappa light chain DPK9/JK1 (SEQ ID NO:65), shark VNAR 5A7 (SEQ ID NO: 66), and a humanized 5A7 variant,5A7-IVabc (SEQ ID NO: 67). The residue numbering refers to E06 sequence;(b) individual amino acid sequences of the humanized VNAR domains of theinvention: 5A7 (5A7 IVabc) (SEQ ID NO: 67), humanized versions of theanti-HSA domain E06 versions 1.1 through to 1.10 and H08 version 1.1(SEQ ID NOs: 68-78); (c) alignment of (SEQ ID NO: 46), humanizedE06v1.10 (SEQ ID NO: 74), and improved versions of the humanized E06v1.10 of the invention (SEQ ID NOs: 79-89).

FIGS. 3A and 3B show: (a) a ribbon model representation of the crystalstructure of E06 in complex with HSA; (b) sequence of E06 (SEQ ID NO:46) highlighting-, CDR1, CDR3, HV2 and HV4 and framework regions.Residues identical to human V kappa framework DPK9 are shown in bold.Residues within 5A of HSA in crystal structures, and involved in variousmodes of interactions with HSA, are marked by arrows. Humanizedsequences v1.1 (SEQ ID NO: 68) and v1.10 (SEQ ID NO: 74) are alsoaligned so illustrate the re-introduction of contact residues in v1.10post crystal dataset.

FIGS. 4A to 4E show ability of E06 to extend the serum half-life of anunrelated domain across three species of PK model; (a) in vivo PKanalyses of E06 and H08 in fusion with 2V as measured via iodination, 2Valone and PEGylated 2V as a PK control; (b) (i) LC-MS measurements ofhalf-life of 2V-E06 and (ii) 2V-E06-2V in a murine model of PK bothintra-venous and subcutaneous administration; (c) E06-2V and 2V-E06 in arat model of PK by intra-venous administration; (d) E06-2V and 2V-E06both intra-venous and subcutaneous administrations in a cynomologusmonkey model of PK with E06-2V also as subcutaneous administration; (e)allometric scaling of 2V-E06 based on data attained and compared tohalf-life of albumin in each species.

FIGS. 5A and 5B show the retention of the therapeutic domains A1 and CC3to still bind target and retain functionality when in fusion to E06; (a)cell neutralization assays showing the ability of anti-mICOSL domains A1and CC3 to still prevent ligand-receptor binding; (b) T-cellproliferation assays showing the ability of A1 and CC3 to retain theability to inhibit T-cell proliferation when in complex with E06 (i)(hFc controls shown in (ii).

The invention will also be further described by way of reference to thefollowing Examples which are present for the purposes of illustrationonly and are not to be construed as being limiting on the invention.

The abbreviations used are:

HSA, BSA, MSA, RSA—human, bovine, mouse and rat serum albumin,respectively; HBS, HEPES-buffered saline; HEL, hen egg lysozyme; CDR,complementarity determining region; CM, conditioned medium; FW,framework region; HV, hypervariable region; HRP, horseradish peroxidase;MME, monomethyl ether; PBS, phosphate-buffered saline; PEG, polyethyleneglycol; RMSD, root mean squared deviation; LC-MS, liquid chromatographymass spectrometry.

EXAMPLE 1

Isolation of human serum albumin-binding shark VNARs

As a prerequisite to this work, a sequence database of approximately1600 VNAR sequences from the Spiny dogfish (Squalus acanthias) werecompiled, aligned and analysed to facilitate the design of primer pairsto capture the immune repertoire. This ensured the subsequent immunephage libraries constructed were as representative as possible andlacked bias toward certain isotypes. Spiny dogfish were immunized withhuman serum albumin and VNAR sequences from a seropositive animal wereisolated and made into a phage display library. The library was rescuedand a solid phase sequential bio-panning strategy against 50 □g/ml, 50g/ml and 0.5 □g/ml HSA was employed. After three rounds several highaffinity, highly similar clones against albumin were isolated (FIG.1(a)). Binding at both pH 7.0 and pH 6.0 (to facilitate lyzosomalrecycling) was incorporated as part of the screening strategy. One ofthe best binders, which reacted to HSA, MSA and RSA at pH 7.0 and pH6.0, was called E06 (FIG. 1(b)).

VNAR clone E06 is 103 residues in length and has low sequence similarityto human variable domain sequences (<30% identity), with the closesthuman germline sequences being from VL6 and VH4 families. E06 sequenceis a typical shark VNAR lacking the CDR2 region of mammalian antibody Vdomains and carrying instead a HV2 stretch in FW2 and also HV4 loop aspart of FW3 sequence. Similar to other Ig molecules, there is a 6-aminoacid CDR1 sequence and relatively short 9-amino acid CDR3 sequence (FIG.1(b)). E06 belongs to a structural type IV of shark VNARs, which isdistinct from better-characterized type I (e.g. 5A7 (Dooley et al, MolImmunol 40(1) 25-33 (2003)) and type II (e.g. PBLA8; a phage-displaylibrary clone from HEL-immunized nurse shark) (Dooley et al, PNAS 103(6)1846-51 (2006))). Type IV VNARs have only 2 canonical Ig domain cysteineresidues (positions 22 and 83 in E06), compared to 6 cysteines in type Iand 4 cysteines in type II.

The immunized library was also screened prior to panning which resultedin the isolation of several more diverse anti-HSA VNAR domains (FIG.1(c)). The response was sufficiently robust that selection was notnecessary as the unselected library showed 16% positive binding to HSA.

EXAMPLE 2

Humanization Strategy for Type I and Type IV VNARs

As an initial strategy the well characterized type I VNAR, 5A7 washumanized. To this end, we selected as a template the human Ig variablelight domain (V01) germline sequence, DPK9, which was consideredstructurally the closest to 5A7 of human Ig variable domain sequences.5A7 was humanized by resurfacing, whereby multiple solvent-exposed aswell as core framework residues of 5A7 were replaced by human DPK9/J01residues (FIG. 2(a)). Specifically, the following structural elementswere replaced in 5A7: FW1 (residues 6-21), FW2/part of HV2 (residues38-47), FW3b (residues 67-82) and FW4 (residues 106-113). All sixcysteine residues of type I VNAR scaffold were retained. The resultingmolecule, which we call 5A7-IVabc, has 60 out of 86 (69.8%) of non-CDRresidues, and 60 out of 112 (53.6%) of all residues identical to DPK9

To further validate the humanization-by-resurfacing approach taken with5A7, we created a series of humanized variants of E06 using 5A7-IVabcmolecule as a guide as well as using the crystal structure to ensurecritical interface residues remained within the humanized variants toretain function of the original domain (FIG. 2(a)). To make humanizedE06 variant 1.1 (huE06 v1.1), 30 residues out of 103 total in E06 werereplaced with DPK9 residues. Specifically, the majority of frameworkresidues: FW1 (residues 6-21), FW2 (residues 38-40), FW3b (residues66-82), and FW4 (residues 99-103) of E06 were made identical to DPK9.The majority of these changes parallel those used to make 5A7-IVabc. Theregions left intact (shark) were first 4 amino-terminal residues, CDR1(residues 28-33) and CDR3 (residues 86-94), HV2 (residues 43-52), FW3aand HV4 (residues 53-65). In huE06 v1.1 molecule, 54 out of 85 (63.5%)of non-CDR residues are identical to human DPK9.

To modify E06 further, mutations toward 5A7-IVabc sequence wereintroduced into HV4 region of v1.1 (K61S and T63S) to make huE06 v1.2.Further DPK9-like changes were made in HV2 region (⁴³SSNKE⁴⁷→⁴³KAPK⁴⁶)(SEQ ID NOs: 99 & 100) to produce huE06 v1.7. These two sets of changes(in HV2 and HV4) were combined to make huE06 v1.3. A derivative of v1.3,which had its N-terminus changed toward DPK9 (¹TRVD⁴ to ¹DIQMT⁵) (SEQ IDNOs: 101 & 102), was made and named huE06 v1.4. We also attempted toredesign E06 by shortening the FW3a/HV4 region; to do that, 5 sharkresidues were deleted and 3 DPK9 residues introduces in this area ofhuE06 v1.3 molecule; in addition, Y55F change was introduced. Theresulting molecule was named huE06 v1.5. Finally, huE06 v1.10 wasderived from v1.1 by restoring ³⁸RKN⁴⁰ shark sequence in FW2 fromDPK9-like ³⁸QQK⁴⁰.

During this step wise process, a total of 10 humanized derivatives ofE06 were designed, expressed and characterized—these sequences arelisted in FIG. 2(b). Additional variants of v1.10 with improvedbiophysical characteristics were constructed using random mutagenesis, aphage display library built from these variants and screened on thebasis of retention to bind HSA. Briefly, hE06v1.10 sequence was clonedinto a phagemid vector and was mutated by error-prone PCR aiming at upto 9 substitutions/VNAR sequence using a GeneMorph II random mutagenesiskit (Agilent Technologies, Santa Clara, Calif.) according to themanufacturer's instructions. The resultant mutational phage displaylibraries were rescued and selected twice using Nunc Maxisorpimmunotubes. Following each pan, two 96-well plates of individualcolonies were picked with a QPix2 XT (Genetix, San Jose, Calif., USA).Binding as monoclonal phage and periprep was evaluated by ELISA. Allsamples were processed with a Perkin Elmer MiniTrak robotic liquidhandling system (Waltham, Mass., USA). Unique clones showing OD450 byperiprep ELISA at least 25% higher than the readings obtained fromparental hE06v1.10 were selected for transfer to a eukaryotic expressionvector. Following preliminary screen, clones exhibiting the best bindingproperties were selected for expression transiently in 1 litre culturesof HEK293 cells and monomeric 6×His tagged VNAR proteins purified byIMAC and cation IEX chromatography. The clones with the best biophysicalpropertied based on HSA binding and no evidence of aggregation arelisted in FIG. 2(c).

EXAMPLE 3

Structure of E06 in Complex with HSA

E06 was expressed as a monomeric 6×His-tagged protein and crystallizedin complex with HSA and the structure was determined to 3.0 Å (FIG.3(a)).

Protocols: Crystallization

E06:HSA crystals were grown by hanging drop vapor diffusion at 18° C. indrops containing 1.0 μl protein stock solution (11.0 mg/ml proteincomplex, 25 mM Tris pH 7.4, 150 mM NaCl) mixed with 1.0 μl well solution(16% PEG 2000 MME, 100 mM sodium acetate pH 4.6) and equilibratedagainst 0.5 ml of well solution. Chunky crystals grew in approximatelyone week, measuring ˜50 μm across.

Data Collection and Processing

E06 complex crystals belong to the space group P3221 with unit cellparameters 127.98×127.98×151.76 Å, and contain two molecules of E06 andtwo molecules of HSA in the asymmetric unit, implying a solvent contentof 54.2%. Crystals were drawn through a solution of 20% DMSO and 80%well solution, and cooled rapidly in liquid nitrogen. Diffraction datawere recorded at APS beamline 22-ID on a MAR-300 detector. Intensitieswere integrated and scaled using the program Xia2.

Phasing, Model Building, and Refinement

The structure of E06 in complex with HSA was determined by molecularreplacement with PHASER using the crystal structure of apo HSA (PDB ID:1AO6) as a starting search model. A few rounds of refinement with Phenixwere performed, after which clear density for the p-sheet regions of EO6was obtained. After subsequent placement of a poly-alanine model of EO6and several iterative cycles of model rebuilding with Coot andrefinement with autoBuster, the final Rwork and Rfree values of 23.73%and 26.63% were obtained. In contrast to the classical antigen-antibodyrecognition mode, it was found that most extensive interactions with HSAoriginate from the CDR3 residues and framework residues on E06 (FIG.3(b)). Antigen binding results in a large buried surface area of 705 Å,which is 12.5% of total surface area of E06. The interaction elucidatedis also unusual for a VNAR due to the planar nature of the interface andinclusion and contribution of framework residues in the antigen-antibodycomplex.

EXAMPLE 4

Functional properties of humanized E06 variants

5A7-IVabc protein showed excellent expression profile in mammalian cellswith very little aggregation in either monomeric or dimeric (with humanFc) format and full retention of binding activity to HEL. The bindingconstant for monomeric VNARs to HEL, as determined by Biacore, was 13.6nM for parental 5A7 and 14.8 nM for humanized 5A7-IVabc (Table 1). Theresults show the retention of binding antigen by humanized VNAR domainsof the invention as measured by BIAcore against target; (Table 1) showsretention of binding of humanised 5A7 to hen egg lysozyme (HEL); (Table2 and 3) shows retention of albumin binding by humanized E06 v1.1 andv1.10 to human, mouse and rat albumin at pH 7.4 and pH 6.0.

TABLE 1 ka kd KD KD Rmax Chi² % surface Sample (1/Ms) (1/s) (M) (nM)(RU) tc (RU²) active 5A7-AAA-6xHis 4.48E+06 6.09E−02 1.36E−08 13.6 61.765.47E+07 0.31 65.0% 5A7IVabc-AAA-6xHis 1.17E+07 1.73E−01 1.48E−08 14.869.67 6.26E+07 0.272 74.1%

E06 and its humanized variants were first expressed as human Fc fusions.The expression levels of the humanized variants, such as 1.1, 1.2, 1.3,1.5 and 1.7, were not dramatically different from the parental E06molecule and were in 10-40 μg/ml range in transient COS-1 system.

To assess the kinetic parameters of binding of E06, huE06 v1.1 and huE06v1.10 to serum albumins, the monomeric (6×His-tagged) V-NARs were testedin BIAcore experiments. As shown in Table 2 (at pH 7.4) and Table 3 (atpH 6.0), the humanized versions retained the ability to bind mammalianalbumin species with nanomolar affinities. Increased affinity of thehumanized versions was seen at the lower pH values compared of wild typeE06.

TABLE 2 ka kd KD Fold change Rmax % Surface Chi² IgNAR Albumin (1/Ms)(1/s) (nM) from E06 (RU) Active (RU²) E06 human 3.48E+06 6.55E−04 0.19n/a 86.18 85.3% 0.81 huE06v1.1 human 8.72E+05 1.40E−02 16.0 84.9 78.677.8% 0.234 huE06v1.10 human 5.59E+05 3.57E−03 6.4 33.9 80.62 79.8%0.181 E06 mouse 2.18E+06 1.80E−03 0.83 n/a 109.9 85.2% 0.871 huE06v1.1mouse 8.41E+05 4.14E−02 49.2 59.4 100.9 78.2% 0.354 huE06v1.10 mouse4.12E+05 9.59E−03 23.2 28.1 100.9 78.2% 0.26 E06 rat 2.21E+06 3.20E−031.45 n/a 93.71 88.4% 0.687 huE06v1.1 rat 4.36E+05 3.29E−02 75.3 52.077.32 72.9% 0.616 huE06v1.10 rat 4.49E+05 1.65E−02 36.8 25.4 84.03 79.3%0.311

TABLE 3 ka kd KD Fold change Rmax % Surface Chi² IgNAR Albumin (1/Ms)(1/s) (nM) from E06 (RU) Active (RU²) E06 human 1.12E+07 1.59E−03 0.14n/a 50.1 49.6% 0.254 huE06 v1.1 human 3.62E+06 1.84E−02 5.07 35.6 29.329.0% 0.196 huE06 v1.10 human 3.52E+06 6.37E−03 1.81 12.7 39.7 39.3%0.334 E06 mouse 9.85E+06 3.23E−03 0.33 n/a 44.1 34.2% 0.259 huE06 v1.1mouse 3.43E+06 3.18E−02 9.28 28.3 21.0 16.3% 0.143 huE06 v1.10 mouse3.14E+06 1.25E−02 3.98 12.2 31.7 24.5% 0.182 E06 rat 1.26E+07 4.33E−030.34 n/a 41.8 39.5% 0.244 huE06 v1.1 rat 3.12E+06 3.10E−02 9.94 29.018.5 17.4% 0.118 huE06 v1.10 rat 3.65E+06 1.52E−02 4.17 12.2 29.1 27.4%0.16

EXAMPLE 5

E06 Extends Plasma Half-Life of Unrelated Fusion Proteins In Vivo

The unrelated naïve VNAR domain, 2V, was identified as a type IV duringthe initial database acquisition of sequences. It has no known bindingpartner and was therefore chosen as a suitable “dummy” protein partnerto study the PK and PD of E06 in several animal models. Molecularfusions of E06 with N-terminal, C-terminal and dual terminal constructswere created using G4S linker sequences bridging the VNAR domains, andC-terminal AAA-6×HIS tags for purification purposes. Dimers and trimerswere expressed in HEK293 cells and purified using standard Ni-NTAmethods and SEC as a final polishing step. Proteins were assessed forrodent viruses and endotoxin levels prior to use in animal models.BIAcore analysis was conducted to determine the affinities of eachfusion variant compared to wild-type E06. Table 4 shows the on, offrates and KD affinity values for wild-type, 2V-E06, E06-2V and 2V-E06-2Vfusions with 2V control and the anti-HEL 5A7 as an additional control.In particular Table 4 shows the BIAcore analyses of E06 alone, as anN-terminal, C-terminal and dual fusion construct with 2V, 2V alone and5A7 as control binding to HAS.

TABLE 4 mean ka ± SE mean kd ± SE mean KD ± SE Sample Ligand (×10⁶ M⁻¹s⁻¹) (×10⁻⁴ s⁻¹) (nM) n E06 HSA 3.092 ± 0.034  5.825 ± 0.103 0.189 ±0.005 4 CSA 2.675 ± 0.023  5.845 ± 0.185 0.219 ± 0.005 2 MSA 2.316 ±0.010 17.130 ± 0.360 0.740 ± 0.012 2 RSA 2.240 ± 0.058 30.110 ± 0.1101.345 ± 0.030 2 HEL — — — 2 E06-2V HSA 7.464 ± 0.384  5.307 ± 0.1470.071 ± 0.002 4 CSA 8.996 ± 0.841  5.915 ± 0.370 0.066 ± 0.002 2 MSA5.299 ± 0.463 14.695 ± 0.415 0.279 ± 0.017 2 RSA 5.553 ± 0.452 27.510 ±0.310 0.498 ± 0.035 2 HEL — — — 2 2V-E06 HSA 0.953 ± 0.097  6.412 ±0.116 0.721 ± 0.095 6 CSA 0.820 ± 0.138  6.821 ± 0.163 0.910 ± 0.157 4MSA 0.862 ± 0.018 21.420 ± 0.150 2.486 ± 0.036 2 RSA 0.623 ± 0.09134.418 ± 0.550 5.949 ± 0.984 4 HEL — — — 2 2V-E06-2V HSA 0.689 ± 0.064 5.762 ± 0.068 0.896 ± 0.091 8 CSA 0.592 ± 0.069  5.904 ± 0.186 1.052 ±0.155 4 MSA 0.507 ± 0.047 19.783 ± 0.473 4.032 ± 0.472 4 RSA 0.448 ±0.041 31.353 ± 0.919 7.245 ± 0.934 4 HEL — — — 4 2V HSA — — — 4 CSA — —— 2 MSA — — — 2 RSA — — — 2 HEL — — — 2 5A7 HSA — — — 2 CSA N.A. N.A.N.A. — MSA — — — 2 RSA N.A. N.A. N.A. — HEL 1.543 ± 0.025 468.450 ±11.350 30.365 ± 0.255  2

The data shows that E06 tolerates bath N-terminal and C-terminal fusionscreating dimers proteins in addition to both N and C terminal fusionconstructs creating trimers. All constructs retain high affinity bindingto target (HSA) and across other albumin species (mouse, rat andmonkey). Note that in Table 4, “-” indicates no binding and N.A. thatthe experiment was not conducted. All three constructs were tested inanimal model of PK to measure the ability of E06 to extend plasmahalf-life of an unrelated partner protein. As an initial study, EOG (SEQID NO: 46) and the related protein H08 (sequence listed in FIG. 1(a) asP2_H08, SEQ ID NO: 36, was isolated from same panning strategy as E06)were fused to 2V and compared to the clearance of 2V alone. PurifiedE06-2V and H08-2V were studied in a murine single dose, PK model todetermine the serum half-life of 2V as an independent domain or incomplex with the anti-HSA VNAR domains. To ensure iodination did notaffect HSA binding, E06-2V and H08-2V were “cold” iodinated showing thatthis did not interfere with E06 binding HSA.

Male C57BL/6 mice were injected i.v. with the following doses ofprotein: 1 mg/kg for 2V and 2V-PEG (2×NOF) and 0.3 mg/kg for the tandemVNARs (E06-2V and H08-2V). The concentrations for tandem VNARs werescaled for 1 mg/kg dosage and the dose for 2V-PEG was based on proteinonly. Radioactive equivalent concentrations were determined bygamma-counting. Individual concentration values <LOQ (defined as3*background cpm) were treated as zero for calculations of the mean andSD; N=6 per time point. Plasma concentration are illustrated in FIG.4(a) and PK parameters summarized in Table 5 which shows PK and PDmeasurements showing increase in half-life of E06 fusion with 2Vcompared to 2V alone.

TABLE 5 AUC_(0-∞) AUC_(0-∞)/Dose Compound Dose C_(max) ^(a) (μg (μg eq.hr/mL)/ CL Vd_(ss) t_(1/2) (Protocol) (mg/kg) (μg eq./mL) eq. hr/mL)(mg/kg) (mL/hr/kg) (mL/kg) (hr) E06 (anti-HSA)-2V 0.3 4.53 47.5 158 6.32203 47.8 tandem (09_2691) H08 (anti-HSA)-2V 0.3 4.81 47.9 160 6.26 17939.7 tandem (09_2992) 2V -PEG (09_2313) 1 17.8 533 533 1.89 95.8 53.5 2V(08_3952) 1 8.44 2.33 2.33 430 869 0.146 (~9 min)

The resultant measured half-life is shown in FIG. 5(b) clearly showingthat E06 and H08 extended the plasma half-life of 2V from approximately9 minutes to 47.8 h and 39.7 h respectively.

As a more precise means of measuring protein clearance and ensuring thatfusion partners are intact, an LC-MS method was carried out measuringpeptides specific to E06 and 2V. For the mouse model, 2V-E06 and2V-E06-2V were injected at a dose of 4 and 2 mg/kg, respectively, bothi.v. and s.c. into groups of 12 CD1 mice. Two blood samples plusterminal bleeds were taken from each animal at intervals to provideduplicate samples to cover time points from 1 h-168 h (FIG. 5(b). Forthe rat model, E06-2V and 2V-E06 were injected i.v. at 1 mg/kg intogroups of 3 wistar rats and blood samples taken from 0.25-168 h. A PKstudy in cynomolgus monkeys was carried out with E06-2V and 2V-E06 dosedat 1 mg/kg i.v. to groups of two animals. Blood sampling was carried outat from 0.25 h-28 days. After 28 days, the 2V-E06 group were injecteds.c. with 0.5 mg/kg protein and sampling carried out over 14 days.

Table 6 summarizes the pharmacokinetic data measured from theseexperiments showing extended half-life of the E06 containing fusions,good correlation between intra-venous and subcutaneous administrationand rapid distribution where pharmacokinetic parameters were measuredacross all three PK models.

TABLE 6 Dose Cl Vdss t½ F Species Construct (mg/kg) Route (ml/hr/kg)(ml/kg) (h) (%) Rat E06-2V 1 iv 3.3 105  22 — 2V-E06 1 iv 2.7 94 25 —Mouse 2V-E06 4 iv 2   96 33 — 2V-E06 4 sc — — 25 48 2V-E06- 2 iv 3.8112  21 — 2V 2V-E06- 2 sc — — 26 46 2V NHP 2V-E06 1 iv  0.25 75 210 —E06-2V 1 iv  0.29 69 164 — E06-2V 0.5 sc — — * >75  * = Not sufficientdata to determine accurate half-life for E06-2V delivered via thesubcutaneous route, although data shown graphically to approximate tointravenous dosing half-life.

The half-life of 2V-E06 fusions across all three species in comparisonto that of albumin was plotted. Overall these data demonstrate that thehalf-life values for 2V-E06 lie within 2 fold of those of albumin in all3 species investigated. However, as all the half-life values obtainedfor 2V-E06 are similar to those of albumin it is believed that theaddition of 2V-E06 has improved the pharmacokinetic of 2V because highaffinity binding of the molecule to albumin has led to the 2V-E06albumin complex taking on the pharmacokinetic and clearance propertiesof albumin.

Assuming that the 2V-E06 albumin complex takes on the pharmacokineticproperties of albumin, the prediction of the likely pharmacokineticproperties of this molecule in human becomes simply a case ofunderstanding the half-life of albumin in human. Literature data isavailable for the half-life of albumin in human (19 days). It istherefore anticipated that the half-life of 2V-E06 in human willapproximate to 19 days and that it volume of distribution willapproximate to 0.1 I/kg (assume volume of distribution is conservedacross species).

For each animal model, VNAR concentrations in plasma were analyzed byquantitative LC-MS as described. Briefly, plasma samples were treated asfollows: 50 μl plasma was added to 50 μl 6 M guanidine containing thepeptide internal standard and reduced with 20 μl of 32 mM Tris(2-carboxy-ethyl) phosphine-hydrochloride (TCEP) at 56° C. for 45minutes. Samples were alkylated by addition of 10 μl of 128 mMiodoacetamide at 37° C. for 60 minutes. Samples were diluted by theaddition of 150 μl 100 mM phosphate, pH8, 0.1% CHAPS. Using a Kingfishermagnetic bead processor, magnetic Ni-beads (25 μl/sample) were washed in100 mM phosphate, pH8, 0.1% CHAPS before being transferred to plasmasample plate and incubated for 1 h. Three washes were carried out: 1stand 2nd wash: transfer beads to plate containing 100 μl phosphate, pH8,0.1% CHAPS; 3rd wash: transfer beads to plate containing 100 μlphosphate, pH8, 0.1% CHAPS+20 mM imidazole. Bound VNAR was then elutedby transferring the beads to a plate containing 100 μl phosphate, pH8,+250 mM imidazole. Beads were removed and 100 μl 100 mM TRIS, pH8containing 20 μg/ml Trypsin was added and incubated for 4 h at 37° C.Following this, 20 μl 100 mM TRIS, pH8 containing 100 μg/ml Trypsin wasadded and incubated overnight at 37° C. Samples were then loaded into aCTC PAL auto-sampler and analysed using LC-MS. Signature peptides wereseparated on an Agilent 1100 HPLC system using an Onyx monolithic RP C18guard trapping cartridge and a Waters XBridge BEH130 C18 Column, 3.5 μM,2.1×100 mm analytical column. Peptides were eluted with a gradient of 5%to 45% acetonitrile in water with 0.1% formic acid. Signature peptideswithin each partner were analysed independently: E06 signaturepeptide—EQISISGR and 2V signature peptide—AQSLAISTR. The analytes weredetected by atmospheric pressure electrospray ionisation MS/MS using anAB Sciex AP15500 QTRAP triple quadrupole mass spectrometer. The ionchromatograms were quantified by reference to standards spiked intofresh control plasma and analysed over the range 0.04 to 50 μg/ml. Theion chromatograms were integrated and quantified by interpolation of thestandard curve with a 1/y weighting using AB Sciex Analyst 1.5.1software.

EXAMPLE 6

Therapeutically Relevant Domains Retain Function when Fused to E06

As 2V had no inherent target antigen or function, it was a good partnerfor in vivo PK work however to determine the ability of E06 not toinhibit nor impair the function of a fused partner protein, anti-ICOSLVNAR domains (A1 and CC3) in fusion with E06 were studied. Both A1 andCC3 have exhibited efficacy in vitro and in vivo and were goodcandidates to validate E06's utility as a domain capable of extendingthe half-life of therapeutically relevant domains.

N, C and N/C terminal fusion of anti-murine ICOSL VNAR (A1 and CC3) andE06 were constructed, expressed, purified and assessed for dual bindingagainst both HSA and mICOSL by BIAcore analyses (Table 7). Allconstructs were immobilized with HSA on the chip, with flowing overmICOSL. Both dimers and trimers of E06 with A1 and CC3 retained theability to bind both HSA and ICOSL (A1-Fc average affinity: KD=6.27×10⁻⁷M; CC3-Fc average affinity: KD=4.96×10⁻⁸ M. 2V did not bind ICOSL).

TABLE 7 Domain KD (M) A1-E06 2.6 × 10⁻⁷ ± 2.5 × 10⁻⁸ E06-A1  2.1 × 10⁻⁷± 3.43 × 10⁻⁸ A1-E06-A1 4.7 × 10⁻⁷ ± 9.0 × 10⁻⁸ CC3-E06 7.7 × 10⁻⁸ ± 1.6× 10⁻⁸ E06-CC3 1.0 × 10⁻⁷ ± 1.7 10⁻⁸   CC3-E06-CC3 1.0 × 10⁻⁷ ± 1.3 ×10⁻⁸ 2V-E06-2V —

To assess the ability of A1 and CC3 to retain the ability to blockligand binding to receptor in cell based neutralization assays, E06fusions were incorporated (FIG. 6(b)) and the IC₅₀ values measured. Theassay was carried out follows: CHO cells expressing murine ICOSLreceptor were grown to confluency in DMEM/F12+5% FBS media in 96-wellcell culture plates (Greiner, Bio-One). mICOSL-hFc (20 μl at 450 ng/ml)was pre-incubated for 1 h with 40 μl of anti-mICOSL-NAR fused to EO6 inDMEM/F12+2% FBS and then added to the cells. Following 1 h incubation at16° C. cells were gently washed 3 times with DMEM/F12+2% FBS andincubated for another 40 min at 16° C. with goat anti-human Fc-HRP(SIGMA) diluted 1:10 000 in the same media. Afterwards the cells werewashed again 3 times with DMEM/F12+2% FBS media and ones with PBS anddeveloped with TMB substrate. The results show that single digit nMefficacy was achieved showing the retention of neutralization by both A1and CC3 when in complex with E06. Results are shown in Table 8 ofBIAcore analyses of fusion domains immobilized with HSA and bindingmICOSL.

TABLE 8 Concentration IC₅₀ IC₅₀ Name mg/ml uM M_(r) ng/ml nm A1-E060.056 2.16 25.9 182.6 7.05 E06-A1 0.078 3.01 25.9 125.3 4.84 A1-E06-A10.077 1.95 39.5 53.81 1.36 CC3-E06 0.039 1.51 25.9 61.18 2.36 E06-CC30.051 1.97 25.9 244 9.42 CC3-E06-CC3 0.043 1.09 39.5 24.45 0.62

As a secondary measurement of functionality, T-cell proliferation assayswere carried out as follows: antibodies were titrated in 96 well TC flatbottom plate in 100 ul assay media (use the media listed above, butleave out the Rat T stim, IL-2 and IL-lalpha). Tosyl activated magneticDynal beads are coated per product insert instructions with hu or muICOSL, anti-mu CD3e and hIgG1 filler (1 ug ICOSL/0.5 ug anti-CD3/3.5 ughIgG1 per 1×10{circumflex over ( )}7 beads). Prior to assay set up,titer beads to determine optimal concentration that gives around8000-40,000 CPM. Generally for hu ICOSL this will be around 40,000beads/well and for mu ICOSL, around 20,000 beads/well. Add 50 μl/well ofthe appropriate beads to the titered antibodies. D10.G4.1 cells arewashed 4× with assay media and resuspended to 8×10{circumflex over ( )}5cells/ml and add 50 μl/well=40,000 cells/well. All wells are brought upto a final volume of 200 μl and incubated for 48 hours. 1 μci/well3H-thymidine is added and incubated for 5-7 hours. Harvest and countCPM. Both A1 and CC3 still retained the ability to inhibit T cellproliferation when in fusion with E06 as shown in Table 9 and Table 10with pM IC₅₀ values.

TABLE 9 Sample IC₅₀ nM A1-E06-A1 24.02 CC3-E06-CC3 5.772V-E06-2V >128.21 Anti-ICOSL control 0.04

TABLE 10 Sample IC₅₀ nM A1-hFc 2.28 CC3-hFC 0.32 Anti-ICOSL control 0.03Rat IgG2A control —

The invention claimed is:
 1. A fusion protein comprising: A) a single domain specific binding molecule comprising the Framework Regions FW1, FW2, FW3a, FW3b, and FW4, in which: i) FW1 comprises TRVDQSPSSLSASVGDRVTITCVLT (SEQ ID NO: 28), ARVDQSPSSLSASVGDRVTITCVLR (amino acids 1-25 of SEQ ID NO: 67) or TRVDQTPRTATRETGESLTINCVLT (SEQ ID NO: 1), ii) FW2 comprises TYWYQQKPGS (SEQ ID NO: 29), TCWYQQKPGK (amino acids 34-43 of SEQ ID NO: 67), or TYWYRKNPGS (SEQ ID NO: 2), iii) FW3a comprises GRYSESVN (SEQ ID NO: 30), or GRYVESVN (SEQ ID NO: 3), iv) FW3b comprises FTLTISSLQPEDFATYYCRA (SEQ ID NO: 31), FTLTISSLQPEDFATYYCGL (amino acids 65-84 of SEQ ID NO: 67) or FSLRIKDLTVADSATYICRA (SEQ ID NO: 4), and v) FW4 comprises GAGTKVEIK (SEQ ID NO: 32), CGQGTKVEIK (amino acids 103-112 of SEQ ID NO: 67) or GAGTVLTVN (SEQ ID NO: 5); or B) a single domain specific binding molecule comprising framework regions FW1, FW2, FW3a, FW3b, and FW4 in which: i) FW1 comprises a sequence having at least 50% identity to TRVDQSPSSLSASVGDRVTITCVLT (SEQ ID NO: 28), ARVDQSPSSLSASVGDRVTITCVLR (amino acids 1-25 of SEQ ID NO: 67) or TRVDQTPRTATRETGESLTINCVLT (SEQ ID NO: 1), ii) FW2 comprises a sequence having at least 50% identity to TYWYQQKPGS (SEQ ID NO: 29), TCWYQQKPGK (amino acids 34-43 of SEQ ID NO: 67, or TYWYRKNPGS (SEQ ID NO: 2), iii) FW3a comprises a sequence having at least 50% identity to GRYSESVN (SEQ ID NO: 30), or GRYVESVN (SEQ ID NO: 3), iv) FW3b comprises a sequence having at least 50% identity to FTLTISSLQPEDFATYYCRA (SEQ ID NO: 31), FTLTISSLQPEDFATYYCGL (amino acids 65-84 of SEQ ID NO: 67) or FSLRIKDLTVADSATYICRA (SEQ ID NO: 4), and v) FW4 comprises a sequence having at least 50% identity to GAGTKVEIK (SEQ ID NO: 32), CGQGTKVEIK (amino acids 103-112 of SEQ ID NO: 67) or GAGTVLTVN (SEQ ID NO: 5).
 2. The fusion protein as claimed in claim 1, which is humanized.
 3. The fusion protein of claim 1, wherein the single domain specific binding molecule has an amino acid sequence selected from the group consisting of: E06 (SEQ ID NO. 46), BB11 (SEQ ID NO. 85) and H08 (SEQ ID NO: 36), or a sequence having at least 50% identity thereto.
 4. The fusion protein of claim 1, wherein the single domain specific binding molecule has an amino acid sequence selected from the group consisting of: huE06 v1.1 (SEQ ID NO. 68), huH08 v1.1 (SEQ ID NO. 75), huE06 v1.2 (SEQ ID NO. 69), huE06 v1.3 (SEQ ID NO. 70), huE06 v1.4 (SEQ ID NO. 71), huE06 v1.5 (SEQ ID NO. 72), huE06 v1.6 (SEQ ID NO. 76), huE06 v1.7 (SEQ ID NO. 73), huE06 v1.8 (SEQ ID NO. 77), huE06 v1.9 (SEQ ID NO. 78), huE06 v1.10 (SEQ ID NO. 74, AC9 (SEQ ID NO. 79), AD4 (SEQ ID NO. 80), AG11 (SEQ ID NO. 81), AH7 (SEQ ID NO. 82), BA11 (SEQ ID NO. 83), BB10 (SEQ ID NO. 84), BC3 (SEQ ID NO. 86), BD12 (SEQ ID NO. 87), BE4 (SEQ ID NO. 88), or BH4 (SEQ ID NO. 89), or a sequence having at least 50% identity thereto.
 5. The fusion protein of claim 1, in which the fusion protein further comprises a biologically active agent fused to the single domain specific binding molecule.
 6. The fusion protein of claim 5 wherein the biologically active agent is a pharmaceutically active agent.
 7. The fusion protein of claim 5, wherein the biologically active agent is a chemical compound or polymer.
 8. The fusion protein as claimed in claim 6, wherein the pharmaceutically active agent is selected from the group consisting of a toxin, anti-inflammatory drug, antibiotic, anti-cancer drug, and analgesic.
 9. The fusion protein as claimed in claim 6, wherein the pharmaceutically active agent is a protein.
 10. The fusion protein as claimed in claim 9, wherein the protein is an enzyme or fragment thereof, or a cytokine or fragment thereof.
 11. The fusion protein as claimed in claim 8, wherein the toxin is cholera toxin or a non-protein toxin.
 12. The fusion protein as claimed in claim 8, wherein the anti-inflammatory agent is a non-steroidal anti-inflammatory drug (NSAID) or steroid.
 13. The fusion protein as claimed in claim 8, wherein the anti-cancer drug is cytotoxic or cytostatic.
 14. The fusion protein as claimed in claim 7, where the chemical compound or polymer is a substance suitable to extend the half-life of the fusion protein in vivo.
 15. The fusion protein as claimed in claim 14, wherein the chemical compound or polymer is polyethylene glycol (PEG) and/or cyclodextrin.
 16. The fusion protein as claimed in claim 5, wherein the biologically active agent is linked via a linker moiety to the single domain specific binding molecule.
 17. The fusion protein as claimed in claim 16, wherein the linker moiety is a peptide, peptide nucleic acid or polyamide linkage.
 18. The fusion protein as claimed in claim 5, wherein the single domain specific binding molecule and biologically active agent are joined together by chemical means.
 19. The fusion protein as claimed in claim 5, wherein the single domain specific binding molecule and biologically active agent are joined together by peptide bonds through protein synthesis.
 20. The fusion protein as claimed in claim 5, wherein the single domain specific binding molecule and biologically active agent are N-, C- and/or N-/C-terminal fusion(s).
 21. A nucleic acid encoding the fusion protein of claim
 9. 22. A nucleic acid construct comprising a nucleic acid as claimed in claim
 21. 23. A host cell comprising a vector as claimed in claim
 22. 24. A process for the production of a fusion protein comprising the step of expressing the nucleic acid sequence of claim 21 in a host cell.
 25. The process as claimed in claim 24, further comprising the step of purifying the expressed fusion protein.
 26. The process as claimed in claim 25, wherein the fusion protein is humanised.
 27. A process for the production of a fusion protein of claim 7, wherein the fusion is by chemical means.
 28. The process as claimed in claim 27, wherein the chemical means include hydrogen bonds, salt bridges, chemical cross-linking or any combination thereof.
 29. A process for the production of a fusion protein comprising the step of expressing the nucleic acid sequence encoding the single domain specific binding molecule of claim 1 in a host cell, and fusing the expressed protein to a chemical compound or polymer.
 30. The process as claimed in claim 29, wherein the single domain specific binding molecule is humanised.
 31. The process of claim 29, wherein the fusion is chemical synthetic or biosynthetic.
 32. The process of claim 31, wherein the biosynthetic fusion is enzymatic.
 33. A pharmaceutical composition comprising a fusion protein as claimed in claim
 1. 34. The pharmaceutical composition as claimed in claim 33, further comprising at least one of a pharmaceutically acceptable carrier, a pharmaceutically acceptable diluent, a pharmaceutically acceptable adjuvant, and a pharmaceutically acceptable buffer solution. 